Remedies for thrombocytopenia

ABSTRACT

Agents for preventing and treating thrombocytopenia, thrombocytopenic purpura and various diseases that tend to cause bleeding due, presumably, to thrombocytopenia contain a parathyroid hormone (PTH) or at least one PTH derivative as an active ingredient.

FIELD OF INVENTION

This invention relates to agents for preventing and treatingthrombocytopenia that contain a parathyroid hormone (PTH) or aderivative thereof as an active ingredient.

BACKGROUND OF INVENTION

Platelets, as well as erythrocytes and neutrophils which are also in theclass of mature corpuscles, originate from hematopoietic stem cells andare produced by the differentiation and proliferation of these cells. Inthe early stage of the hematopoietic process, hematopoietic stem cellsdifferentiate and proliferate to megakaryocytes via their precursorcells. Mature magakaryocytes form proplatelets which would eventually bereleased into peripheral blood as platelets. It has been found thatvarious hematopoietic factors and cytokines are involved in the seriesof steps in the above-described platelet production process. Forexample, it has been shown experimentally that interleukin-3 is involvedin the stage from stem cells to megakaryocytes, interleukin-6 in thematuring of megakaryocytes and thrombopoietin in the stage from stemcells to the maturing of megakacyocytes. It is speculated that a certainfactor is also involved in the process of platelet production andrelease from mature magakacyocytes but no such factors are yet to beidentified.

Thrombocytopenia manifests itself if either one of the steps in theabove-described thrombpoletic process is interfered with and there aretwo major causes of such interference, abnormality in hematopoieticcells and abnormality in hematopoietic factors. In the first case, thedifferentiation and proliferation of hematopoietic cells are interferedwith by either congenital or acquired causes. Aplastic anemia andosteomyelodysplasia syndrome are known to occur by congenital causes andbone marrow transplantation and the administration of chemotherapeuticsare two known acquired causes. As for the abnormality in hematopoieticfactors, cyclic thrombocytopenia is known to be caused by this reason.

Transfusion of platelets is currently considered to bean effective meansof treating thrombocytopenia but this treatment does not necessarilysupply an adequate amount of platelets and, in addition, it involves therisk of infection with viruses and other pathogens. Therefore, thedevelopment of agents effective in preventing and treatingthrombocytopenia is desired.

A parathyroid hormone (PTH) is known as one of the important hormonesfor bone metabolism. Numerous reports have so far been published todescribe the actions of PTH on the bone but there are few reports on itsaction on the hematopoietic system. The exception is the report byMeytes et al. (J. Clin. Invest. Vol. 67, 1263-1269:1981), which showedthat PTH interfered with the in vitro colony formation by BFU-E(precursor cells of erythrocytes) and CFU-GM (precursor cells ofgranulocytes and macrophages). Thus, nothing has been unravelled aboutthe action of PTH on thrombopoiesis.

SUMMARY OF INVENTION

An object of the present invention is to provide agents for preventingand treating thrombocytopenia. Another object of the invention is toprovide pharmaceutical drugs that are effective in treating orpreventing diseases that accompany thrombocytopenia.

As the result of their intensive studies, the present inventors foundthat a parathyroid hormone (PTH) or its derivatives are effective in thetreatment of thrombocytopenia, and the present invention has beenaccomplished on the basis of this finding.

Thus, the present invention relates essentially to an agent forpreventing and treating thrombocytopenia that contains a parathyroidhormone (PTH) or a derivative thereof as an active ingredient. Theinvention also relates to an, agent for preventing and treatingthrombocytopenia that contains human PTH(1-84) or one or more derivatiesthereof as an active ingredient. The invention further relates to anagent for preventing and treating thrombocytopenia that contains aparathyroid hormone (PTH) or human PTH(1-34) as an active ingredient.The invention also relates to an agent for preventing and treatingthrombocytopenia that contains a parathyroid hormone (PTH) as an activeingredient. The invention further relates to an agent for preventing andtreating thrombocytopenia that contains human PTH(1-84) as an activeingredient. The invention also relates to an agent for preventing andtreating thrombocytopenia that contains a derivative of parathyroidhormone (PTH) as an active ingredient. The invention further relates toan agent for preventing and treating thrombocytopenia that containshuman PTH(1-34) as an active ingredient. It should be noted that asidefrom thrombocytopenia, the agents of the invention for preventing andtreating thrombocytopenia are intended to prevent and treat variousdiseases that are attributable to thrombocytopenia such asthrombocytopenic, purpura and various diseases that tend to causebleeding due, presumably, thrombocytopenia.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the platelet increasing action of PTH (whenadministered in a single dose to mouse);

FIG. 2 is a graph showing the platelet increasing action of PTH (whenadministered daily to mouse);

FIG. 3 is a graph showing the platelet increasing action of a PTHderivative (when administered daily to mouse); and

FIG. 4 is a graph showing the platelet increasing action of PTH (whenadministered daily to rabbit)

DETAILED DESCRIPTION OF THE INVENTION

The parathyroid hormone (PTH) to be used in the invention may occur invarious forms including PTH of a, native type, PTH produced by geneticengineering techniques and PTH synthesized chemically; preferably itrefers to human PTH(1-84) composed of 84 amino acid residues. Examplesof PTH derivatives include partial peptides of the PTH as defined above,the constituent amino acids of the PTH per se of partial peptidesthereof which are partly replaced by other amino acids, the constituentamino acids of the PTH per se or partial peptides thereof which arepartly depleted, as well as peptides that have at least amino acid addedto the PTH per se or partial peptides thereof; the peptides as PTHderivatives should have similar activities to the PTH itself. Exemplarypartial peptides of PTH include human PTH(1-34), human PTH(1-64),)humanPTH(35-84) and bovine PTH(1-34). PTH(1-34)refers to a partial peptide ofPTH that is composed of 34 amino acids as counted from the N terminus ofPTH.

Preferred examples of amino acid replacement include substitution ofleucine or norleucine for the constituent amino acid in the 8-position,substitution of leucine or norleucine for the constituent amino acid inthe 18-position, and substitution of tyrosine for the constituent aminoacid in the 34-position.

Preferred examples of the parathyroid hormone (PTH) or its derivativeswhich are to be used as agents for preventing and treatingthrombocytopenia in the invention include human PTH(1-84), humanPTH(1-34), human PTH(1-38), human PTH(1-37), human PTH(1-34)—NH₂ and soforth; more preferred examples include human PTH(1-84) and humanPTH(1-34); the most preferred is human PTH(1-84).

The agents for preventing and treating thrombocytopenia refer to thetherapeutics and preventives, preferably therapeutics, ofthrombocytopenia, therombocytopenic purpura and various diseases thattend to cause bleeding due, presumably, to thrombocytopenia. Conditionsincluded in the scope of thrombocytopenia are the disease caused byradiation therapy and the disease accompanying bone marrowtransplantation. Also included in the scope of thrombocytopenia are thedisease caused by the selective suppresion of megakaryocytes due, forexample, to pharmaceutical drugs (phenylbutazone, gold compounds,tolbutamide and chemotherapeutics) and viral infection, the diseasescaused by systemic myelocytic deficiencies such as aplastic anemia,autoimmune thrombocytopenic purpura, osteomyelodysplasis syndrome,leukemia, mutiple myeloma and megakaryoblastic anemia, drug-inducedimmune thrombocytopenia, post-transfusion purpura and secondary immunethrombocytopenia.

A typical dosage form for the pharmaceutical drugs of the invention isas an injection (e.g. liquid preparations and lyophilized preparations)that is produced by ordinary pharmaceutical formulation proceduresapplicable to peptides; also useful are dosage forms that are intendedto be show local and delayed actions, as by inclusion withinmicrocapsules or incorporation in sheets of gel. When formulatingpharmaceutical preparations, pharmaceutically acceptable adjuvants maybe added. In order to increase the half-time in blood, pharmaceuticalpreparations modified with polyethylene glycol may be formulated.

Useful adjuvants include base materials, stabilizers, antiseptics,preservatives, emulsifiers, suspending agents, solubilizers, solventpromoters, lubricants, flavoring agents, coloring agents, fragrances,soothing agents, vehicles, binders, viscous agents, buffers, etc.Specific examples include calcium carbonate, lactose, sucrose, sorbitol,mannitol, starches, amylopectin, cellulosic derivatives, gelatin, cacaobutter, distilled water for injections, aqueous sodium chloride,Ringer's solution, glucose solution, human serum albumin and so forth.

When preparing the pharmaceutical drugs of the invention using theabove-mentioned adjuvants, suitable adjuvants may be selected withreference to a certain data book such as “A List of PharmaceuticalAdditives” (published by the Committee on Medical Laws and Regulations,Tokyo Society of Pharmaceutical Industry, Foundation and by the StudyCommittee on Medical Laws and Regulations, Osaka Society ofPharmaceutical Industry, Foundation). The amounts in which the adjuvantsare to be used my be selected as appropriate for the dosage form andother factors from within the ranges tolerated by pharmaceuticalformulation procedures.

The pharmaceutical drugs of the invention may be administered by eithersystemic or local route, preferably by a local route such assubcutaneously, intravenously, intranasally or transpulmonarily. Inprinciple, the duration of administration should not be shorter than theperiod over which the patient is clinically diagnosed as suffering fromthrmobocytopenia; depending on the etiology of the disease, the clinicalphysician may decide to continue the administration after the patient'srecovery. If the occurrence of thrombocytopenia is anticipated as in thecase where a chemotherpeutic is being administered, the drugs of theinvention may be administered for preventive purposes even if thepatient is not currently suffering from disease. The frequency ofadministration may range from once a month to daily administration;preferably, the frequency ranges from once in two weeks to about fivetimes a week or, alternatively, the drugs may be administered daily.

The dose of the PTH of the invention varies with the disease it isindicated for, the severity of the disease and other factors; forsystemic administration, the PTH's dose ranges from about 1 μg to about1000 μg per kg of body weight, preferably from 5 μg to 200 μg per kg ofbody weight.

EXAMPLES

The present invention will now be described in greater detail withreference to the following examples. The human PTH(1-84) used inExamples 1-4 was prepared by an improved version of the methodsdescribed in Japanese Domestic Announcement (kohyo) No. 505259/1992 andJ. Biol. Cheml., 265, 15854(1990). The human PTH(1-34) used in Example 3was purchased from Peptide Institute, Inc.

Example 1 Test for Single-dose Administration to Mouse

(Animal on Experiment)

Nine-week old male C57Bl/6N mice as purchased from Japan Charles RiverCo. Ltd. were used in the experiment. (Preparation of Drug Solutions tobe Administered) Human PTH(1-84) was dissolved in a citrate buffer(pH-5; containing 0.05% of Tween 80) at final concentrations of 1, 0.2and 0.04 μg/mL so as to prepare the drug solutions to be administered.

(Administration)

Each of the thus prepared drug solutions and a control (citrate buffer)was administered to 15 mice through the tail vein in a volume of 10mL/kg. Hence, the dose of the human PTH(1-84) was 0, 0.4, 2 and 10μg/kg, respectively. The administration was initiated at 10 a.m.

(Blood Sampling and Counting peripheral Corpuscle)

After 3, 6 and 9 hours of the administration, five mice were picked upat random from each group of animals and blood samples were withdrawnthrough the orbital vein. At 10 a.m. on the same day, separate bloodsamples were taken from untreated mice (four in number) to obtain thepre-administration value. Peripheral corpuscle counts were obtained withan automatic corpuscle counter F-800 (TOA MEDICAL ELECTRONICS CO., LTD.)

(Results)

The results are shown in FIG. 1. The group administered with 10 μg/kg ofPTH showed a significant increase in platelet counts over the control at6 and 9h after the administration. Thus, the platelet count increasedwithin a short period after the administration of PTH.

Example 2 Test for Daily Administration to Mouse

(Animal on Experiment)

Nine-week old male C57Bl/6N mice as purchased from Japan Charles RiverCo., Ltd. were used in the experiment.

(Preparation of Drug Solutions to be Administered)

Drug solutions to be administered were prepared basically by the sameprocedure as in Example 1 to give drug concentrations of 5, 20 and 80μg/wL.

(Administration)

The mice under test were divided into four groups, which wereadministered subcutaneously with the three drug solutions and a control(citrate buffer) once a day in a volume of 10 mL/kg. Hence, the dose ofthe drug was 0, 50, 200 and 80 μg/kg, respectively. The administrationwas initiated at 10 a.m.

(Blood Sampling and Counting Peripheral Corpuscles) On days 5, 7 and 9after the start of administration, five or six mice were picked up atrandom from each group of animals and blood samples were withdrawnthrough the orbital vein. The sampling of blood was timed to occur 24 hof the final administration. Separate blood samples were taken fromuntreated mice (five in number) to obtain the value for normal mouse.Peripheral corpuscle counts were obtained with an automatic corpusclecounter F-800 (TOA MEDICAL ELECTRONICS CO., LTD.)

(Results)

The results are shown in FIG. 2. On day 5, the platelet count showed atendency to increase only in the groups administered 200 and 800 μg/kgof PTH but on day 7, all of the treated groups were significantly higherin platelet count than the control. It was therefore clear that the PTHalso had a platelet increasing action even when it was administereddaily.

Example 3 Effectiveness of PTH Derivative

(Animal on Experiment)

Ten-week old male C57Bl/6N mice as purchased from Japan Charles RiverCo., Ltd. were used in the experiment.

(Preparation of Drug Solutions to be Administered)

Drug solutions to be administered were prepared basically from humanPTH(1-84) and human PTH(1-34) by the same procedure as in Example 1 togive a drug concentration of 20 μg/mL.

(Administration)

Eighteen mice were divided into three groups, which were administeredsubcutaneously with the two drug solutions and a control (citratebuffer) for six days on a once-a-day basis in a volume of 10 mL/kg.Hence, the dose of the drug was 0 and 200 μg/kg, respectively. Theadministration was initiated at 10 a.m.

(Blood Sampling and Counting Peripheral Corpuscles)

After 24 h of the final administration, a blood sample was withdrawnfrom each mouse through the orbital vein. Peripheral corpuscle countswere obtained with an automatic corpuscle counter F-800 (TOA MEDICALELECTRONICS CO., LTD)

(Result)

The results are shown in FIG. 3. Obviously, PTH(1-34) has a comparableplatelet increasing action to PTH(1-84), From these results, one maywell expect that even shorter partial peptides of PTH as well as partialpeptides of intermediate sizes, for example, PTH(35-84) and PTH(1-64)would have similar actions.

Example 4 Test for Daily Administration to Rabbit

(Animal on Experiment)

Six 12-15 week old male Japanese albino rabbits JW/CSK were used in theexperiment. The animals were individually kept in aluminum rabbitbracket cages Rb-1 (350^(w)×500^(d)×350^(h) mm) in a vavarium in aspecific pathogen-free environment under the following conditions:temperature, 24±2° C.; humidity, 50±10%; illumination, light turned onat 5:00 and turned off at 19:00; ventilation, 15 changes/hr. Each animalwas supplied with 120 g of a radiation-sterilized feed for rabbits RM(purchased from Funahashi Farm) per day. The animals were also allowedto drink to tap water ad libitum from an automatic water feeder.

(Preparation of Drug Solutions to be Administered)

Human PTH(1-84) was administered in a volume of 2 mL/vial at aconcentration of 200 μg/mL. A solvent was also supplied in a volume of 2mL/vial. Both the drug and the control were stored at −135° C. untiluse.

(Method of Administration)

PTH (200 μg/kg) was administered to the rabbits subcutaneously at theback of their neck for 13 consecutive days. The solvent was similarlyadministered in the same volume as the dose of a rh-PTH solutioncalculated from their body weight.

(Blood Sampling and Counting Peripheral Corpuscles)

Blood samples (0.5 mL) were withdrawn from the rabbits through thelateral auricular vein before the administration and on days 2, 6, 9 and13 during the administration period. Each blood sample was distributedamong blood testing vessels SB-41S (TOA MEDICAL ELECTRONICS CO., LTD.)and heparinized; thereafter, platelet counts were obtained with anautomatic corpuscle counter F-800 (TOA MEDICAL ELECTRONICS CO. LTD.)

(Results)

Three rabbits were daily administered with 200 μg/kg of PTH andplatelets were counted on days 0, 2, 6, 9 and 13 of the administrationperiod. The results are shown in FIG. 4. Starting on day 2 of theadministration, the platelet count increased until day 6, exhibiting twoto four times as great as the initial value. The platelet countsubstantially returned to the initial level on day 13.

INDUSTRIAL APPLICABILITY

The agents of the present invention which contain a parathyroid hormone(PTH) or a derivative thereof as an active ingredient are useful astherapeutics and preventives of thrombocytopenia, thrombocytopenicpurpura and various diseases that tend to cause bleeding due,presumably, to thrombocytopenia

What is claimed is:
 1. A method for treating thrombocytopenia in apatient in need thereof, comprising administering to said patient aneffective amount of a composition comprising parathyroid hormone (PTH)or a derivative thereof for a time and under conditions to amelioratesaid thrombocytopenia.
 2. The method according to claim 1, wherein humanPTH (1-84) is administered.
 3. The method according to claim 1, whereinhuman PTH (1-34) is administered.
 4. The method according to claim 1wherein said effective amount is in the range of 1 μg to 1,000 μg per kgof body weight administered systematically at a frequency ranging fromonce per day to once per month.
 5. The method according to claim 1wherein said effective amount is from 5 μg to 200 μg per kg of bodyweight administered from once every two weeks to once daily.
 6. Themethod according to claim 1 wherein the route of said administration issubcutaneously.
 7. The method according to claim 1 wherein the route ofsaid administration is intravenously.
 8. The method according to claim 1wherein the route of said administration is intranasally.
 9. The methodaccording to claim 1 wherein the route of said administration istranspulmonarily.
 10. The method of claim 1 wherein said PTH or PTHderivative is selected from the group consisting of human PTH (1-34),human PTH (1-64), human PTH (35-84), bovine PTH (1-34), human PTH(1-84), human PTH (1-38) and human PTH (1-37).
 11. The method accordingto claim 1 wherein said PTH or PTH derivative modified with polyethyleneglycol and administered.
 12. The method according to claim 1 whereinsaid PTH or PTH derivative is administered encapsulated withinmicrocapsules.
 13. The method according to claim 1, wherein said PTH orPTH derivative is incorporated in a sheet of gel.